Plant Chromosome-Specific Probes by Microdissection of a Single Chromosome: Is That a Reality?

Abstract

Painting plant chromosomes through chromosomal in situ suppression (CISS) hybridization has long been considered impracticable. Seeking to build specific and complex probes from a single microdissected chromosome, we employed human chromosomes as models to standardize all the necessary steps for application in plants. Human metaphases were used to define the adequate conditions for microdissection, chromosome DNA amplification and labeling through degenerate oligonucleotideprimed PCR, and in situ hybridization stringency. Subsequently, these methodologies were applied in the plant species Zea mays (chromosome 1) and Capsicum annuum (chromosome 7 or 8). The high quality of human and plant cytogenetic preparations and the meticulous standardization of each step, especially the most critical onesmicrodissection and first round of DNA amplification -were crucial to eliminate the signs of non-specific hybridization and for direct application in plants. By overcoming these challenges, we obtained chromosome-specific probes, which allowed to achieve a clear and uniform painting of the entire target chromosomes with little or no background, evidencing their complexity and specificity. Despite the high amount of ubiquitous repetitive sequences in plant genomes, the main drawback for chromosome painting, we successfully employed our methodology on two plant species. Both have more than 80% repetitive sequences, which is compared to the human genome (66-69%). This is the first time that plant chromosome-specific probes were successfully obtained from a single A mitotic or meiotic microdissected chromosome. Thereby, we assume that chromosome painting through microdissection and CISS hybridization can now be considered a reality in the field of plant cytogenetics.

The Kengyilia hirsuta karyotype polymorphisms as revealed by FISH with tandem repeats and single-gene probes

“…This implies that the Btr-1 and Btr-2 might be more strongly conserved in the 3St and 3P chromosomes. Moreover, gene cloning may be facilitated by genome dissection during flow sorting or micro dissection (Tiwari et al 2015;Said et al 2018;Sheng et al 2020;Soares et al 2020). In this study, the chromosomes belong to the homoeologous group 3 were clearly identified.…”

“…Alien gene transfer by interspecific hybridization is strongly affected by chromosome collinearity (Friebe et al 1996;Qi et al 2007). Targeted gene cloning may be facilitated by capturing the syntenic chromosomes of the wild relatives, which include complex genomes, by flow sorting (Tiwari et al 2015;Said et al 2018) or even by micro dissection (Sheng et al 2020;Soares et al 2020).…”

Cadmium‐mediated toxicity in plant cells is associated with the DCD/NRP‐mediated cell death response

“…The integrity and concentration of the amplification product were evaluated on 1.5% agarose gel and NanoDrop™ Spectrophotometer. GmNAC81 probes (~700 bp) were generated by a second round of the PCR reaction under the same conditions, adjusting the dTTP concentration to 100 μM and adding 40 µM of the nucleotide Tetramethyl‐rhodamine 5‐dUTP (Roche®) for probe labelling (Soares et al, 2020). GmNAC81 copy number was determined from the fluorescent signals obtained by in situ hybridisations (nuclei‐FISH) performed on the slides containing isolated and preserved nuclei of the BR16, NAC81.1 and NAC81.3 lines.…”

“…GmNAC81 copy number was determined from the fluorescent signals obtained by in situ hybridisations (nuclei‐FISH) performed on the slides containing isolated and preserved nuclei of the BR16, NAC81.1 and NAC81.3 lines. The slides were pretreated with 1× PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 , pH 7.4) for 5 min, 4% formalin for 10 min, 1× PBS for 5 min, and cold ethanol series of 70%, 85% and 100% for 5 min each, and the nuclear chromatin was denatured in 70% formamide/2× SSC buffer (3M NaCl, 0.3M Na 3 C 6 H 5 O 7 .2H 2 O, pH 7.0) for 3 min at 67°C and dehydrated in 70%, 85% and 100% cold ethanol series (Schwarzacher & Heslop‐Harrison, 2000; Soares et al, 2020). The hybridisation mix consisted of 50% formamide, 2× SSC, and 200 ng of the probe, followed by its denaturation in a thermocycler for 5 min at 85°C.…”

Antioxidants and antigenotoxic properties of Rhizopus oligosporus fermented cassava (Manihot esculenta Crantz)

“…After pressing two root tips onto each slide for 10 min, they were coloured with acetocarmine and cover slips were carefully attached to exclude air bubbles. The cover slips were sealed on the slides with clear fingernail polish as suggested by Soares et al (2020). This is to prevent drying out of the preparation by the heat of the microscope (Sharma, 1983).…”